Studies of enzyme immunoassay and related methods (EIA) show that a series of enzymes is known which are suited as markers (G. B. Wisdom: Clin. Chem. 22 (1976) 1242-1255; A. H. W, M. Schuurs, B. K. van Weemen (1977) in: Enzyme immunoassay, Grundlagen und Praktische Anwendung, Fundamentals and Practical Application), p. 4-9, Georg Thieme Verlag Stuttgart 1978; H. Keller: Medizine. Laboratorium 31 (1978), 83-94; T. Porstmann, S. T. Kiessig: J. Immunol. Methods 150 (1992) 5-21). However, to date horseradish peroxidase (HRP) is the choice enzyme because it is comparatively cheap, very pure and can be, therefore, produced with a high specific enzyme activity, and it is a "robust" biosubstance allowing chemical reactions without essentially loosing the reactivity of enzymes. For this reason, commercially available HPR conjugates are used in nearly 80% of all enzyme immunoassays. In the remaining approx. 20% mainly conjugates with an alkaline phosphatase are used. Only in special cases, labeling is effected with other enzymes such as e.g. .beta.-galactosidase, glucose oxidase or acetylcholine esterase etc.
In preparing an enzyme conjugate the enzyme is crosslinked by a covalent bond with the immune substance to be labeled, without a reduction of neither the enzyme activity nor the immunoreactivity of the antigen or antibody. This presupposes that the two coupling partners have functional groups where appropriate coupling reagents can act directly or that reactive groups such as e.g. thiol or bismaleinimide groups are introduced and then in a second reaction step the conjugation is indirectly effected by homo- or heterobifunctional reagents (E. Ishkawa et al.: J. Immunoassay 4 (1983) 209-327). Already in the early 60 efforts have been made to produce HRP conjugates as markers for immunohistochemical examinations and as EIA tracers. According to the developments in this field at that time e.g. (i) 4,4'-difluoro 3,3'-dinitrophenyl sulfone (J. S. Ram: Biochim. Biophys. Acta 78 (1963) 228-230; P. K. Nakane, G. B. Pierce: J. Histochem. Cytochem. 14 (1966) 929), (ii) various carbodimides (S. Avrameas, J. Uriel: C. R. Acad. Sci. (D) Paris 262 (1966) 2543; P. K. Nakane, G. B. Pierce: J. Cell Biol. 33 (1967) 307-318), (iii) cyanuric chloride and bisdiazotized o-dianisidine (S. Avrameas: Bull. Chim. Biol. 50 (1968) 1169) etc. are used for the direct coupling of HRP and immunoglobulins (IgG). However, only IgG homopolymers are formed as main products of these so-called "one-pot processes", and most of the time not more than 5% of the HRP used will be obtained in the form of the desired HRP-IgG conjugate.
It was detected that the cause for this unexpected reaction behavior of HRP was that a molecule of the commercial horseradish peroxidase has only one to two reactive amino groups in spite of its comparatively high molecular weight of approx. 40 000. When isolating the enzyme from the plant by allyl isothiocyanate, forming of the substances ascorbic acid and sinigrin contained the majority of the originally available .alpha.- and .epsilon.-NH2 groups will be blocked. (L. Ornstein: J. Histochem. Cytochem. 14 (1966) 929; K. G. Welinder, L. B. Smillie, G. R. Schonbaum: Can. J. Biochem. 50 (1972) 44-62). In the direct crosslinking of HRP with immunoglobulins by glutaric dialdehyde described in 1969, which is carried out under mild reaction conditions through .epsilon.-amino groups of Lysine (S. Avrameas: Immunochemistry 6 (1969) 43) only about 5 to 10% coupling is obtained. Intensive self-crosslinking of the immunoglobulin takes place with high-molecular heterogeneous conjugates being formed (A. H. Korn, S. H. Feairheller, E. M. Filachione: J. Mole. Biol. 65 (1972) 525; D. H. Clyne, S. H. Norris, R. R. Modesto et al.: J. Histochem. Cytochem. 21 (1973) 233). Although it was found that the enzyme activity was essentially not affected, it was also found that when using this coupling method the immunoreactivity clearly declined (D. M. Boorsa, G. L. Kalsbeck: J. Histochem. Cytochem. 23 (1975) 200). Nevertheless, in the early 70 numerous HRP immunoconjugates were prepared for EIA purposes, primarily by this one-step glutaric aldehyde method.
In 1971 we succeeded in improving this one-step glutaric aldehyde coupling method (S. Avrameas, T. Ternynck: Immunochemistry 8 (1971) 1175-1179) after detecting that an HRP molecule is able to react only with one molecule of glutaric dialdehyde due to the small number of reactive amino groups even if the coupling reagent is available at an excess. The second aldehyde group cannot react with the same or another HRP molecule. This peculiar reaction behavior of HRP provides the basis for the so-called two-step coupling method in which the enzyme is first induced to react solely with glutaric dialdehyde. After separating the excess of the coupling reagent a monomeric HRP-glutaric dialdehyde coupling product is obtained, like some "activated peroxidase", which can react with the primary NH.sub.2 groups of an antigen or antibody in a second reaction step in which preferably monomeric conjugates are formed (S. Avrameas: Histochem. J. 4 (1972) 321). Conjugates are also formed which contain 2 moles of enzyme per IgG molecule (D. M. Boorsma, J. G. Streefkerk: J. Immunol. Methods 30 (1979) 245-255). When applying this method of conjugation the reactivity of HRP is reduced by 30 to 50%, yet the loss of immunoreactivity is smaller than when applying the one-step method (T. J. Greenwalt, E. McF.Swierk, E. A. Steaner: J. Histochem. Cytochem. 21 (1973) 233), the efficiency of enzyme cross-linkage becomes insignificantly higher (N. Yamamoto: Acta Histochem. Cytochem. 8 (1975) 41). In spite of the aforementioned disadvantages a number of HRP conjugates were prepared as EIA tracers by this method (B. K. Weeman, A. H. W. M. Schuurs: FEBS Lett. 15 (1971) 232: Avrameas, B. Guilbert: Biochimie 54 (1972) 837).
The conditions for preparing the enzyme conjugates were still not satisfactory in the early 70 in spite of the use of enzyme immunological detection methods. This was the possible reason for Nakane and Kawoi developing a completely new strategy for preparing HRP-labeled antibodies in 1974. They proceeded on the basis that most of the enzymes were glycoproteins. In the case of horseradish peroxidase carbohydrates total approx. 18% of its molecular weight, and consist of 8 carbohydrate chains of known composition arranged on the surface of the enzyme, yet they are not related to the enzymatic activity of the molecule (L. Shannon, E. Kay, J. Y. Lew: J. Biol. Chem. 241 (1966) 2166-2172; K. G. Welinder, L. B. Smillie: Can. J. Biochem. 50 (1972) 63-90; K. G. Welinder: Eur. J. Biochem. 96 (1979) 483-509). If horseradish peroxidase is oxidized with sodium metaperiodate, the hydrocarbon residues are split with vicinal OH groups and aldehyde groups being formed without a significant loss of enzymatic activity (P. K. Nakane, A. Kawoi: J. Histochem. Cytochem. 22 (1974) 1084-1091). In this manner an "activated" HRP is formed which can directly react with primary NH.sub.2 groups of an antigen or antibody, forming Schiff's bases. Subsequently, the unsaturated azomethine binding is appropriately stabilized by hydration with sodium borohydride. The HRP-labeled tracer is separated in a manner known per se by gel filtration or dialysis.
The coupling method adopted by Nakane avoids difficulties that could occur in earlier conjugation methods due to the fact that an HRP molecule has only one 50th or even fewer reactive primary amino groups which can be affected by the coupling reagents compared to an IgG molecule. As no IgG self-crosslinking will take place, the coupling efficiency is high: it totals approx. 70% as to HRP, and 90-95% as to IgG. When applying this method neither the enzyme activity nor the immunoreactivity are fully maintained. The corresponding data in literature vary, they closely depend from the reaction conditions of the sodium metaperiodate oxidation and the reduction with NaBH.sub.4. As the oxidized HRP molecule has a bigger number of glucose hemiglutaryl linkers on its surface, it is in a position to conjugate not only with one, but with several IgG molecules. Depending on the stoichiometric ratio the oxidized HRP and IgG are converted to form crosslinked aggregates of a molecular weight of 400 000 and more. The few primary amino groups still available in the HRP molecule were blocked with 1-fluoro-2,4-dinitrobenzene before the NaIO.sub.4 oxidation took place to exclude crosslinking of HRP molecules.
After detailed examination of the effect of individual stages of the NaIO.sub.4 coupling on the enzymatic and the immunological properties of the HRP IgG conjugates, Wilson and Nakane published an improved method in 1978 which formed the basis for peroxidase labeling until today (B. Wilson, P. K. Nakane: Recent Developments in the Periodate Method of Conjugating Horseradish Peroxidase to Antibodies, Immunofluorescence and Related Staining Techniques (Eds.: W. Knapp, K. Holubar, G. Wick), pp. 215-224, Elsevier: North-Holland Biomedical Press 1978). Blocking of the primary NH.sub.2 groups with 1-fluoro-2,4-dinitrobenzene was considered essential before the NaIO.sub.4 oxidation was stopped. In spite of this precaution self-crosslinking of the oxidized HRP could not be completely suppressed as the protective blocking had to take place in an alkaline buffer solution, and sodium metaperiodate is partly inactivated in an alkaline medium. Therefore, oxidation of HRP is suitably conducted in a neutral or weak acetic acid solution. For that reason the periodate excess is separated by dialysis or gel filtration at a pH between 4 and 5. Even if the HRP activated by the formed aldehyde groups is stored for a short time, self-conjugation will remain under 5% at this low pH. Optimum reaction conditions prevail if the 1st stage of the Wilson-Nakane coupling method, the oxidation of HRP, is carried out in the dark at a NaIO.sub.4 concentration of 0.02 M at room temperature, and a 200 times molar peroxidase excess of 200. Thus, a photochemical oxidation of the hydrocarbon residues by ozone formed during photodecomposition of NaIO.sub.4 is avoided (E. B. Dikova, E. M. Gavrilova, A. M. Yegorov: Bioorgan. Khim. (Moscow) 16 (1990) 476-481). A sufficient reaction time is 20 minutes; there are groups which reduce it even to 10 minutes (M. Imagawa, S. Yoshitake, Y. Hamaguchi et al.: J. appl. Biochemistry 4 (1982) 41-57). Wilson and Nakane mention that an HRP molecule oxidized in this manner with NaIO.sub.4 has at least 18 reactive aldehyde catcher groups at its surface, however, not all of them can be used for coupling with IgG molecules, presumably for steric reasons. The 2nd stage of the coupling, the conjugation of the oxidized HRP with immunoglobulin or other proteins which are to be labeled by horseradish peroxidase, is carried out in a solution buffered by bicarbonate at pH&gt;9, optimally between 9.5 and 9.8 while forming Schiff's bases. Wilson and Nakane point out that the coupling time should not be less than 2 hours at room temperature. More favorably the reaction is to be allowed to take place within 4 hours at room temperature. Multi-component mixtures of oligomeres of a differing molecular mass are formed here depending on the stoichiometric ratio of the reactants. If a clear excess of oxidized HRP is used, 5-6 HRP molecules can be bound to an IgG molecule without steric hindrance. The formation of polymeric conjugates is unavoidable since an oxidized HRP molecule can react again with more than one IgG molecule by its aldehyde groups. A model describing this complex reaction process is in good agreement with the experimental results obtained by Wilson and Nakane was prepared by Archer (P. G. Archer: Biometrics 32 (1976) 369-375). Under higher reaction conditions the experimental values are below the values calculated according to the model. They are also disturbing by a high error rate in the spectrophotometric determination of the peroxidase share of a conjugate measured at 403 nm, and of its total protein content resulting from the extinction at 280 nm.
The 3rd stage of the Wilson-Nakane coupling method, the hydration with sodium borohydride, is beyond any doubt a critical stage which requires stabilization of the unsaturated azomethine bonds of the Schiff's bases and also the conversion of the excessive aldehyde groups into the respective carbinols through reduction. This reaction is effected immediately after coupling. It is carried out in a solution buffered by bicarbonate at pH 9.5 at 4.degree. C., with NaBH.sub.4 used in a molar excess of 100 times the HRP that is used. The reaction time should be not less than 2 hours. Though NaBH.sub.4 is a selective reducing agent, various groups mentioned that its use results in a remarkable loss of enzyme activity which can total 18% and more. For this reason, Pierce, a U.S. company recommends to dispense with the stabilization of the azomethine bonds and to quench only the excessive aldehyde groups with Lysine or ethanolamine (Pierce Immuno-Technology Catalog & Handbook 1992) or to use the less strong reactant sodium cyanoborohydride instead of NaBH.sub.4 to show a better compatibility also as compared to IgG (Pierce Seminar 5: Antibody-Enzyme Conjugates, Methods for Preparation and Purification 1993).
The last stage of the Wilson-Nakane coupling method is the isolation of the high-molecular conjugate of the coupling mixture. The separation of the low-molecular coupling products, the starting protein not labeled by HRP and the free enzyme, is an essential step in obtaining a HRP tracer with optimum properties resulting in a remarkable increase of the sensitivity of the assay. This separation task cannot be completed by dialysis or gel filtration, and separation of the tracer in an exclusion volume of a column filled with Sephadex G-25, but rather by gel permeation chromatography through a Sephacryl-S-300.RTM. column (Pharmacia) which is eluted with 0.05 M of PBS buffer (0.15 M of NaCl) at pH 7.4.
In summary, the preparation of a HRP-IgG conjugate according to the Wilson-Nakane method will result in a high-molecular complex where the immunoglobulin molecule represents something like the core which can bind as many HRP molecules on its surface through its activated aldehyde groups as the steric conditions will allow. Presumably, the HRP molecules will be crosslinked among each other only to a minor extent. Sensitive tracers used for an EIA require, however, that the degree of HRP substitution be not less than 2, but it should be clearly higher. Yet, with an increasing degree of substitution the surface of the central immunoglobulin will be increasingly screened off. This is a possible, at least partial explanation for the decline of the observed immunoreactivity. Also the spectrophotometrically obtained faulty total protein content values at 280 nm, can be caused due to the described structure of the HRP conjugate.